Rna viruses for immunovirotherapy

ABSTRACT

The present invention relates to a recombinant virus of the family Paramyxoviridae, comprising at least one expressible polynucleotide encoding a multispecific binding polypeptide, said multispecific binding polypeptide comprising a first binding domain binding to a surface molecule of an immune cell with antitumor activity, preferably a lymphocyte, more preferably a T cell or a dendritic cell, and a second binding domain binding to a tumor-associated antigen; to a polynucleotide encoding the same, and to a kit comprising the same. Moreover, the present invention relates to a method for treating cancer in a subject afflicted with cancer, comprising contacting said subject with a recombinant virus of the family Paramyxoviridae of the invention, and thereby, treating cancer in a subject afflicted with cancer.

The present invention relates to a recombinant virus of the familyParamyxoviridae, comprising at least one expressible polynucleotideencoding a multispecific binding polypeptide, said multispecific bindingpolypeptide comprising a first binding domain binding to a surfacemolecule of an immune cell with antitumor activity, preferably alymphocyte, more preferably a T cell or a dendritic cell, and a secondbinding domain binding to a tumor-associated antigen; to apolynucleotide encoding the same, and to a kit comprising the same.Moreover, the present invention relates to a method for treating cancerin a subject afflicted with cancer, comprising contacting said subjectwith a recombinant virus of the family Paramyxoviridae of the invention,and thereby, treating cancer in a subject afflicted with cancer.

BACKGROUND

Oncolytic viruses (OV) which replicate selectively in tumor cells are anemerging modality of cancer treatment. Aside from direct cytopathiceffects and lysis of tumor cells, interactions of OV with the immunesystem can trigger systemic anti-tumor immunity. OV have been modifiedto express immunomodulatory transgenes to further enhance these effects(Melcher et al., Mol Ther. 2011, 19: 1008-1016). The vaccinia virusJX-594 and herpesvirus talimogene laherpavec (TVEC), both harboringGM-CSF, have shown promising results in clinical phase II and III trials(Heo et al., Nat Med. 2013, 19: 329-336 and Andtbacka et al. J ClinOncol. 2013, 31, suppl; abstr LBA9008).

RNA viruses, in particular members of the family Paramyxoviridae like,e.g. measles virus, have also shown potential use in oncolysis. Virusesof the family Paramyxoviridae are negative-sense single-stranded RNAviruses and include human pathogens like, e.g. human parainfluenzaviruses, mumps virus, human respiratory syncytial virus, and measlesvirus. From wild type measles virus, several non-pathogenic strains,including a vaccine strain, have been derived, which have been shown toremain oncolytic. The measles virus vaccine strain has been developed asa vector platform to target multiple tumor entities and several clinicaltrials are ongoing (Russell et al., Nat Biotechnol. 2012, 30: 658-670).Recently, the capacity of oncolytic MV encoding GM-CSF to support theinduction of a specific anti-tumor immune response in terms of a tumorvaccination effect was demonstrated (Grossardt et al. Hum Gene Ther.2013, 24: 644-654.).

In general, immune response via T cell activation involves theintegration of numerous signals. In order to improve cellular immunityto tumor cells, a variety of immunomodulatory molecules were developed,e.g. “bispecific T cell-engagers” (“BiTEs”). BiTEs are bispecificantibodies, structurally based on two single-chain variable fragments(scFv) with one binding domain targeting the T cell receptor-associatedmolecule CD3 on T cells and the other domain targeting cell surfacemolecules on tumor cells. Such crosslinking of even resting T cells totarget cells induces an artificial immunological synapse and triggers Tcell-mediated target cell lysis. Hence, the BiTE-directed killing isindependent of TCR specificity, costimulation and antigen presentation.

There is, however, still a need in the art for improved cancertherapies, in particular for improved oncolytic viruses.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a recombinant virus of thefamily Paramyxoviridae, comprising at least one expressiblepolynucleotide encoding a multispecific binding polypeptide, saidmultispecific binding polypeptide comprising

a) a first binding domain binding to a surface molecule of an immunecell with antitumor activity, andb) a second binding domain binding to a tumor-associated antigen.

The terms “virus” and “virus of the family Paramyxoviridae” are known tothe skilled person. Preferably, the virus of the family Paramyxoviridaeis a member of the genus Morbillivirus. More preferably, the virus ofthe family Paramyxoviridae is a measles virus (MV), still morepreferably an MV strain Edmonston A or B, or, most preferably, vaccinestrain Edmonston B.

The term “recombinant virus”, as used herein, relates to a viruscomprising a genome modified by biotechnological means as compared toknown, naturally occurring, virus genomes. Preferably, the recombinantvirus is a virus comprising a genome modified as compared naturallyoccurring virus genomes. Preferred biotechnological means for modifyinga viral genome are known to the skilled person and include any of themethods of molecular cloning, in particular recombinant DNA techniquesincluding, without limitation, cleavage of DNA by restriction enzymes,ligation of DNA, polymerase chain reaction (PCR), cloning of viralgenomes, and the like. It is understood by the skilled person thatviruses of the family Paramyxoviridae have a single-stranded (−)-RNA asa genome. Accordingly, the genome of the recombinant virus of thepresent invention, preferably, is obtained by cloning an expressionvector as described herein below comprising an expressible nucleotidesequence encoding said recombinant virus genome, followed by expressingsaid expressible nucleotide sequence encoding said recombinant virus ina permissive host cell. Alternatively, the recombinant virus genome mayalso be expressed in non-permissive host cells, e.g., preferably, fromrodents or other higher eukaryotes.

As used herein, the term “multispecific binding polypeptide” relates toa polypeptide binding, preferably specifically binding, to at least twonon-identical epitopes, wherein said epitopes, preferably, are epitopesof non-identical molecules of interest. Preferably, the multispecificbinding polypeptide is a polypeptide binding, preferably specificallybinding, to two non-identical epitopes, wherein said epitopes,preferably, are epitopes of two non-identical molecules of interest;i.e., the multispecific binding polypeptide, preferably, is a bispecificbinding polypeptide. The multispecific binding polypeptide comprises atleast a first binding domain binding to a surface molecule of an immunecell with antitumor activity. The term “immune cell with antitumoractivity”, as used herein, preferably, relates to a lymphocyte, morepreferably a lymphocyte with the capacity to inactivate cancer cellsand/or to activate cells inactivating cancer cells. More preferably, animmune cell with antitumor activity is a T cell, a dendritic cell or anatural killer cell. Thus, preferably, the multispecific bindingpolypeptide comprises a first binding domain binding, more preferablyspecifically binding, to a surface molecule of a T cell, to a surfacemolecule of a dendritic cell, and/or to a surface molecule of a naturalkiller cell, and a second binding domain binding to a tumor-associatedantigen. Preferably, the multispecific binding polypeptide furthercomprises a transport signal, in particular a peptide export signal.Preferably, the constituent parts of the multispecific bindingpolypeptide, i.e. in particular the first binding domain and the secondbinding domain, are contiguous in amino acid sequence; thus, themultispecific binding polypeptide is, preferably, expressed from asingle open reading frame comprised in a polynucleotide. Thus,preferably, the multispecific binding polypeptide as described hereinabove is a polypeptide expressible from a single transcription unit.Accordingly, preferably, the multispecific binding polypeptide is apolypeptide or a fusion polypeptide. More preferably, the multispecificbinding polypeptide comprises at least one, more preferably two,single-chain antibodies, single-chain Fab polypeptides, or nanobodies.Also preferably, the multispecific binding polypeptide is a secretedpolypeptide.

The term “binding domain” is known to the skilled person and,preferably, relates to a, contiguous or non-contiguous, subpart of apolypeptide having the activity of binding, preferably specificallybinding, to a molecule of interest (cognate antigen). Preferably, thebinding domain binds to its cognate antigen with sufficient affinity toallow detection of a binding domain/antigen complex. Preferably, thedissociation constant (K_(d)) of the binding domain/antigen complex isat most 10⁻⁶ mol/L, more preferably at most 10⁻⁷ mol/L, even morepreferably, at most 10⁻⁸ mol/1, most preferably, at most 10⁻⁹ mol/L. Theterm “specifically binding” is understood by the skilled person.Preferably, specific binding relates to a binding in which the affinityof the binding domain to the cognate antigen is at least threefold, morepreferably at least fivefold, still more preferably at least tenfold,even more preferably at least 100 fold, most preferably at least 1000fold higher than for any non-cognate antigen present in a sample.Accordingly, the dissociation constant (Kd) of any bindingdomain/non-cognate antigen complex is at least 10⁻⁶ mol/L, morepreferably, at least 10⁻⁵ mol/1, most preferably, at least 10⁻⁴ mol/L.

Preferably, the binding domains of the multispecific binding polypeptideare independently selected from the list of molecule types consisting ofa peptide aptamer, an anticalin, a Designed Ankyrin Repeat Protein(DARPin), and an antibody. Preferably, at least one, more preferably twobinding domains are antibodies or parts thereof as specified hereinbelow, more preferably, are single-chain antibodies or nanobodies.

In the context of this invention, a “peptide aptamer” is a peptidespecifically binding a molecule of interest. Peptide aptamers,preferably, are peptides comprising 8-80 amino acids, more preferably10-50 amino acids, and most preferably 15-30 amino acids. They can e.g.be isolated from randomized peptide expression libraries in a suitablehost system like baker's yeast (see, for example, Klevenz et al., CellMol Life Sci. 2002, 59: 1993-1998). The peptide aptamer, preferably, ispresent as a binding domain of the multispecific binding polypeptide. Asused herein, the term “anticalin” relates to an artificial polypeptidederived from a lipocalin specifically binding to a molecule of interest.Similarly, a “Designed Ankyrin Repeat Protein” or “DARPin”, as usedherein, is an artificial polypeptide comprising several ankyrin repeatmotifs and specifically binding a molecule of interest.

As used herein, the term “antibody” relates to a soluble immunoglobulinfrom any of the classes IgA, IgD, IgE, IgG, or IgM, having the activityof specifically binding a molecule of interest. Antibodies againstantigens can be prepared by well known methods using, e.g., a purifiedmolecule of interest or a suitable fragment derived therefrom as anantigen. A fragment which is suitable as an antigen may be identified byantigenicity determining algorithms well known in the art. Suchfragments may be obtained either from one of the molecules of interestby proteolytic digestion, may be a synthetic peptide, or may be obtainedby recombinant expression. Preferably, a peptide of a molecule ofinterest used as an antigen is located at the exterior of a cellexpressing the molecule of interest; i.e. preferably, the epitope thebinding domain interacts with, preferably, is an extracellular domain.Suitability of an antibody generated as a binding domain can be testedby the assay as described herein in the Examples. Preferably, theantibody of the present invention is a monoclonal antibody, a human orhumanized antibody or primatized, chimerized or fragment thereof so longas they exhibit the desired binding activity as specified elsewhereherein. Also comprised as antibodies of the present invention are abispecific antibody, a synthetic antibody, or a chemically modifiedderivative of any of these. Preferably, the antibody of the presentinvention shall specifically bind (i.e. does not cross react with otherpolypeptides or peptides) to a molecule of interest as specified above.Specific binding can be tested by various well known techniques.Antibodies or fragments thereof can be obtained by using methods whichare described, e.g., in Harlow and Lane “Antibodies, A LaboratoryManual”, CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies canbe prepared by the techniques originally described in Köhler andMilstein, Nature. 1975. 256: 495; and Galfré, Meth. Enzymol. 1981, 73:3, which comprise the fusion of mouse myeloma cells to spleen cellsderived from immunized mammals.

“Antibody fragments” comprise a portion of an intact antibody, in anembodiment, comprising the antigen-binding region thereof. Examples ofantibody fragments and fusion proteins of variable regions include Fab,Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; single-domain-antibodies (VHH), alsoknown as nanobodies, and multispecific antibodies formed from antibodyfragments. Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)2fragment that has two antigen-combining sites and is still capable ofcross-linking antigen. “Fv” is the minimum antibody fragment whichcontains a complete antigen-binding site. Preferably, a two-chain Fvspecies consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv (scFv)species, one heavy- and one light-chain variable domain can becovalently linked by a flexible peptide linker such that the light andheavy chains can associate in a “dimeric” structure analogous to that ina two-chain Fv species. It is in this configuration that the threehypervariable regions (HVRs, also referred to as complementaritydetermining regions (CDRs)) of each variable domain interact to definean antigen-binding site. Collectively, the six HVRs of one scFv conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. The term “diabodies”refers to antibody fragments with two antigen-binding sites, whichfragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) in the same polypeptide chain (VH-VL).By using a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies may be bivalent or bispecific. Diabodies are describedmore fully in, for example, EP 0 404 097; WO 1993/01161; Hudson et al.,Nat. Med. 9 (2003) 129-134; and Hollinger et al., PNAS USA 90 (1993)6444-6448. Triabodies and tetrabodies are also described in Hudson etal., Nat. Med. 9 (2003) 129-134.

Preferably, the multispecific binding polypeptide comprises furtheramino acids which may serve e.g. as immunogenic antigens, as a tag forpurification or detection or as a linker. In another preferredembodiment of the multispecific binding polypeptide of the presentinvention, said multispecific binding polypeptide further comprises adetectable tag. The term “detectable tag” refers to a stretch of aminoacids which are added to or introduced into the multispecific bindingpolypeptide of the invention. Preferably, the tag shall be added C- orN-terminally to the multispecific binding polypeptide of the presentinvention. The said stretch of amino acids shall allow for detection ofthe multispecific binding polypeptide by an antibody which specificallyrecognizes the tag or it shall allow for forming a functionalconformation, such as a chelator or it shall allow for visualization byfluorescent tags. Preferred tags are the Myc-tag, FLAG-tag,poly-His-tag, HA-tag, GST-tag or GFP-tag. These tags are all well knownin the art. More preferably, the multispecific binding polypeptidefurther comprises a cytokine as specified elsewhere herein.

Preferably, the multispecific binding polypeptide as described hereinabove is a polypeptide expressible from a single transcription unit.Accordingly, preferably, the multispecific binding polypeptide is apolypeptide or a fusion polypeptide. More preferably, the multispecificbinding polypeptide comprises at least one, more preferably two,single-chain antibodies, single-chain Fab polypeptides, or nanobodies.

Preferably, the first binding domain of the multispecific bindingpolypeptide binds to a surface molecule of a T cell. Preferably, thesurface molecule of a T cell is selected from the group consisting ofCD3, CD2, CD5, CD6, CD9, CD11A, CD25 (IL-2 receptor alpha-chain), CD26,CD28, CD29, CD40L, CD43, CD44, CD45RO, CD45RA, CD45RB, CD47, CD58(LFA-3), CD69, CD70, CXCR4, CD107a, CD122 (IL-2 receptor beta-chain),CD132 (IL-2 receptor gamma-chain), CD134, CD137 and CD247. Morepreferably, the surface molecule of a T cell is CD3. More preferably,the first binding domain comprises a single-chain antibody against CD3.Most preferably, the first binding domain comprises the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2.

Also preferably, the first binding domain of the multispecific bindingpolypeptide binds to a surface molecule of a dendritic cell. Preferably,the surface molecule of a dendritic cell is selected from the groupconsisting of CD1a/b, CD11c, CD16a, CD40, CD68, CD80, CD83, CD86, IFNAR1(interferon-alpha/beta receptor), CD119 (interferon-gamma receptor 1),CDB197 (CCR7), CD205 (DEL-205), CD209 (DC-SIGN) and CD227 (MUC1).

Also preferably, the first binding domain of the multispecific bindingpolypeptide binds to a surface molecule of a natural killer cell (NKcell). Preferably, the surface molecule of a natural killer cell isselected from the group consisting of CD16a, NKG2D, and NCRs such asNKp30, NKp44 and NKp46.

Also preferably, the second binding domain of the multispecific bindingpolypeptide binds to a tumor-associated antigen. Preferably, thetumor-associated antigen is a tumor-associated antigen exposed on thesurface of a tumor cell. More preferably, the tumor-associated antigenis selected from the group consisting of androgen receptor (AR), BCL-1,calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial celladhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogenreceptor (mERs), FAP, HER2/neu, human high molecularweight-melanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21,MOC-52, melan-A/MART-1, melanoma-associated antigen, mucin, OKT9,progesterone receptor (PGR), prostate specific antigen (PSA), prostatestem cell antigen (PSCA), prostate-specific membrane antigen (PSMA),symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33. Most preferably,the tumor-associated antigen is carcinoembryonic antigen (CEA) or CD20.Preferably, the second binding domain comprises a single-chain antibodyagainst CEA. More preferably, the second binding domain comprises theamino acid sequence of SEQ ID NO:3. Also preferably, the second bindingdomain comprises a single-chain antibody against CD20. More preferably,the second binding domain comprises the amino acid sequence of SEQ IDNO:4. Also preferably, the tumor-associated antigen is a glycoprotein ofan oncotropic and/or oncolytic virus.

The term “secreted”, as used herein, relates to a compound beingtransferred from the interior of a host cell to the exterior of saidhost cell by a mechanism intrinsic to said host cell. Preferably, incase the multispecific binding polypeptide is a peptide or polypeptide,said secretion is mediated by a, preferably eukaryotic, signal peptidemediating import of said peptide or polypeptide into the lumen of theendoplasmic reticulum and, more preferably, by the absence of retentionsignals. Signal peptides causing secretion of peptides or polypeptidesare known in the art. Preferably, the signal peptide is or comprises anIg leader sequence. More preferably, the signal peptide is or comprisesa human Ig leader sequence. More preferably, the signal peptide is orcomprises a matching leader sequence, i.e. a leader sequence selectedfrom the same Ig kappa subgroup as the variable light chain of theantibody, preferably, of the single-chain antibody.

The term “cytokine” is known to the skilled person and relates to anyone of a group of peptides released by cells and affecting the state orbehaviour of other or the same cells. Preferably, the cytokine is achemokine, an interferon, an interleukin, a lymphokine, or a tumornecrosis factor. More preferably, the cytokine is GM-CSF (Genbank AccNO: AAA52121.1 GI:181146, preferably encoded by Genbank Acc NO: M10663.1GI:181145) or Interleukin-12 (p35 subunit, Genbank Acc NO: AAD16432.1GI:4323579; p40 subunit, Genbank Acc NO: AAG32620.1 GI:11192035.)

Moreover, also encompassed are variants of the aforementionedmultispecific binding polypeptides. Such variants have at least the sameessential biological activity as the specific multispecific bindingpolypeptides. Moreover, it is to be understood that a variant asreferred to in accordance with the present invention shall have an aminoacid sequence which differs due to at least one amino acid substitution,deletion and/or addition, wherein the amino acid sequence of the variantis still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% identical with the amino sequence of the specificinhibitory peptides. The degree of identity between two amino acidsequences can be determined by algorithms well known in the art.Preferably, the degree of identity is to be determined by comparing twooptimally aligned sequences over a comparison window, where the fragmentof amino acid sequence in the comparison window may comprise additionsor deletions (e.g., gaps or overhangs) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment. The percentage is calculated by determining, preferably overthe whole length of the peptide, the number of positions at which theidentical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison may be conductedby the local homology algorithm of Smith and Waterman (1981), by thehomology alignment algorithm of Needleman and Wunsch (1970), by thesearch for similarity method of Pearson and Lipman (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,PASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by visualinspection. Given that two sequences have been identified forcomparison, GAP and BESTFIT are preferably employed to determine theiroptimal alignment and, thus, the degree of identity. Preferably, thedefault values of 5.00 for gap weight and 0.30 for gap weight length areused. Variants referred to above may be allelic variants or any otherspecies specific homologs, paralogs, or orthologs. Moreover, thevariants referred to herein include fragments of the specificmultispecific binding polypeptides or the aforementioned types ofvariants as long as these fragments and/or variants have the essentialbiological activity as referred to above. Such fragments may be or bederived from, e.g., degradation products or splice variants of themultispecific binding polypeptides. Further included are variants whichdiffer due to posttranslational modifications such as phosphorylation,glycosylation, ubiquitinylation, sumoylation or myristylation.

The term “expressible polynucleotide”, as used herein, relates to apolynucleotide operatively linked to at least one expression controlsequence causing transcription of the nucleic acid sequence comprised insaid polynucleotide to occur, preferably in eukaryotic cells or isolatedfractions thereof, preferably into a translatable mRNA or into a viralgenome. Regulatory elements ensuring expression in eukaryotic cells,preferably mammalian cells, are well known in the art. They, preferably,comprise regulatory sequences ensuring initiation of transcription and,optionally, poly-A signals ensuring termination of transcription andstabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Preferably,the aforesaid at least one expression control sequence is an expressioncontrol sequence of a (−)strand RNA virus, more preferably of aParamyxovirus as described herein above, most preferably of an MV. Thus,preferably, at least one expression control sequence comprises a(−)strand RNA viral regulatory sequence ensuring initiation oftranscription (consensus “gene start signal”, preferably consensus MV“gene start signal”) and termination signals (consensus “gene stopsignal”, preferably, consensus MV “gene stop signal”) ensuringtermination of transcription and stabilization of the transcript. It isknown in the art that production of viral particles in permissive hostcells can be initiated by transfecting into said permissive host cellsone or more expressible DNA constructs encoding (i) a recombinant viralanti-genome, (ii) the viral L gene, (iii) the viral P gene and (iv) theviral N gene. It is also understood by the skilled person that, once aviral genome and the aforesaid viral genes were expressed in said hostcell, replication and assembly of viral particles occurs in thecytoplasm of the host cell and is, therefore, solely dependent on viralregulatory signals. Preferably, the expressible polynucleotide comprisesthe nucleic acid sequence of SEQ ID NO:5.

The term “polynucleotide”, as used in accordance with the presentinvention, encompasses variants of the aforementioned specificpolynucleotides. Moreover, it is to be understood that the polypeptideshaving amino acid sequences of the polypeptides of the present inventionmay also be encoded due to the degenerate genetic code by more than onespecies of polynucleotide. The polynucleotide variants, preferably,comprise a nucleic acid sequence characterized in that the sequence canbe derived from the aforementioned specific nucleic acid sequences by atleast one nucleotide substitution, addition and/or deletion whereby thevariant nucleic acid sequence shall still encode a peptide orpolypeptide having the activity as specified herein. Variants alsoencompass polynucleotides comprising a nucleic acid sequence which iscapable of hybridizing to the aforementioned specific nucleic acidsequences, preferably, under stringent hybridization conditions. Thesestringent conditions are known to the skilled worker and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989),6.3.1-6.3.6. A preferred example for stringent hybridization conditionsare hybridization conditions in 6× sodium chloride/sodium citrate (=SSC)at approximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ depending on the type of nucleic acidand, for example when organic solvents are present, with regard to thetemperature and concentration of the buffer. For example, under“standard hybridization conditions” the temperature differs depending onthe type of nucleic acid between 42° C. and 58° C. in aqueous bufferwith a concentration of 0.1 to 5×SSC (pH 7.2). If organic solvent ispresent in the abovementioned buffer, for example 50% formamide, thetemperature under standard conditions is approximately 42° C. Thehybridization conditions for DNA:DNA hybrids are preferably for example0.1×SSC and 20° C. to 45° C., preferably between 30° C. and 45° C. Thehybridization conditions for DNA:RNA hybrids are preferably, forexample, 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and 55°C. The abovementioned hybridization temperatures are determined forexample for a nucleic acid with approximately 100 bp (=base pairs) inlength and a G+C content of 50% in the absence of formamide. The skilledworker knows how to determine the hybridization conditions required byreferring to textbooks such as the textbook mentioned above.Alternatively, polynucleotide variants are obtainable by PCR-basedtechniques such as mixed oligonucleotide primer-based amplification ofDNA, i.e. using degenerate primers against conserved domains of thepolypeptides or peptides of the present invention. Conserved domains ofthe polypeptides or peptides of the present invention may be identifiedby a sequence comparison of the nucleic acid sequence of thepolynucleotide or of the amino acid sequence of the polypeptides asspecified above. Suitable PCR conditions are well known in the art. As atemplate, DNA or cDNA from appropriate cells may be used. Further,variants include polynucleotides comprising nucleic acid sequences whichare at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% or at least 99% identical to the nucleicacid sequences detailed above. The percent identity values are,preferably, calculated over the entire amino acid or nucleic acidsequence region. A series of programs based on a variety of algorithmsis available to the skilled worker for comparing different sequences asdescribed herein above. A polynucleotide comprising a fragment of any ofthe aforementioned nucleic acid sequences and encoding a polypeptide orpeptide comprising or consisting of the domains conferring thebiological activities of a polypeptide of the present invention is alsoencompassed as a polynucleotide of the present invention. Thepolynucleotide of the present invention shall be provided, preferably,either as an isolated polynucleotide (i.e. isolated from its naturalcontext) or in genetically modified form. The polynucleotide,preferably, is DNA including cDNA, or RNA. The term encompasses singleas well as double stranded polynucleotides. Also included by the termpolynucleotide, preferably, are chemically modified polynucleotidesincluding naturally occurring modified polynucleotides such asglycosylated or methylated polynucleotides or artificially modified onessuch as biotinylated polynucleotides. The polynucleotides of the presentinvention either essentially consist of the aforementioned nucleic acidsequences or comprise the aforementioned nucleic acid sequences. Thus,they may contain further nucleic acid sequences as well.

The term “polynucleotide encoding a recombinant virus”, as used herein,relates to a polynucleotide comprising a nucleic acid sequence ornucleic acid sequences required for generating a virus particle or avirus-like particle in a host cell. It is understood by the skilledperson that a virus is constituted by a polynucleotide genome and atleast one kind of capsid polypeptide. Accordingly, the polynucleotideencoding a recombinant virus of the present invention, preferably,comprises a recombinant virus genome. As will be understood by theskilled person, in case the polynucleotide encoding a recombinant virusis comprised in a virus according to the present invention, thepolynucleotide is (−)strand RNA. It is also understood by the skilledperson that in case the polynucleotide is DNA comprised in a host cell,at least an RNA-dependent RNA polymerase activity will additionally berequired to produce viral particles from said DNA polynucleotide.Preferably, the polynucleotide encoding a recombinant virus comprises orconsists of the nucleic acid sequence of SEQ ID NO:6-9. As annotatedherein, the sequence of the DNA copy of negative-strand (−)RNA virusesis annotated in the usual 5′→3′-orientation; this corresponds to theviral sequence in antigenomic (+)RNA orientation with respect to thenatural 3′→5′-orientation of negative-strand (−)RNA viruses.

As used herein, the term “host cell” relates to a vertebrate cell.Preferably, the cell is a mammalian cell, more preferably, a mouse, rat,cat, dog, hamster, guinea pig, sheep, goat, pig, cattle, or horse cell.Still more preferably, the host cell is a primate cell. Most preferably,the host cell is a human cell. Preferably, the host cell is a tumorcell, more preferably a cancer cell.

Advantageously, it was found in the work underlying the presentinvention that measles virus can be engineered to express multispecificbinding polypeptides destined for secretion and that these polypeptidesare efficiently secreted during viral replication in the cell. Moreover,it was found that by administering measles virus expressing amultispecific binding polypeptide according to the invention, T cellscan be effectively tethered to tumor cells. In contrast to methods ofthe prior art, no systemic treatment with the multispecific bindingpolypeptide is required.

The definitions made above apply mutatis mutandis to the following.Additional definitions and explanations made further below also applyfor all embodiments described in this specification mutatis mutandis.

The present invention further relates to a polynucleotide encoding therecombinant virus of the family Paramyxoviridae according to the presentinvention.

The present invention also relates to a medicament comprising therecombinant virus of the family Paramyxoviridae of the present inventionand at least one pharmacologically acceptable excipient.

The terms “medicament” and “pharmaceutical composition”, as used herein,relate to the compounds of the present invention and optionally one ormore pharmaceutically acceptable carrier, i.e. excipient. The compoundsof the present invention can be formulated as pharmaceuticallyacceptable salts. Acceptable salts comprise acetate, methyl ester, HCl,sulfate, chloride and the like. The pharmaceutical compositions are,preferably, administered locally, topically or systemically. Suitableroutes of administration conventionally used for drug administration areoral, intravenous, or parenteral administration as well as inhalation. Apreferred route of administration is intra-tumoral administration.However, depending on the nature and mode of action of a compound, thepharmaceutical compositions may be administered by other routes as well.For example, polynucleotide compounds may be administered in a genetherapy approach by using viral vectors or viruses or liposomes.

Moreover, the compounds can be administered in combination with otherdrugs either in a common pharmaceutical composition or as separatedpharmaceutical compositions wherein said separated pharmaceuticalcompositions may be provided in form of a kit of parts. The compoundsare, preferably, administered in conventional dosage forms prepared bycombining the drugs with standard pharmaceutical carriers according toconventional procedures. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation. It will be appreciated that the form andcharacter of the pharmaceutically acceptable carrier or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The excipient(s) must be acceptable in the sense of being compatiblewith the other ingredients of the formulation and being not deleteriousto the recipient thereof. The excipient employed may be, for example, asolid, a gel or a liquid carrier. Exemplary of solid carriers arelactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary of liquidcarriers are phosphate buffered saline solution, syrup, oil such aspeanut oil and olive oil, water, emulsions, various types of wettingagents, sterile solutions and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. The diluent(s) is/are selected so as notto affect the biological activity of the combination. Examples of suchdiluents are distilled water, physiological saline, Ringer's solutions,dextrose solution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, non-immunogenic stabilizers and the like.

A therapeutically effective dose refers to an amount of the compounds tobe used in a pharmaceutical composition of the present invention whichprevents, ameliorates or treats the symptoms accompanying a disease orcondition referred to in this specification. Therapeutic efficacy andtoxicity of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician andother clinical factors; preferably in accordance with any one of theabove described methods. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment. A typical dose can be, for example, in the rangeof 1 to 1000 μg for a polypeptide or polynucleotide, or 10⁴-10⁸ viralparticles for a virus or a virus-like particle; however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. Progress can be monitored by periodicassessment. The pharmaceutical compositions and formulations referred toherein are administered at least once in order to treat or ameliorate orprevent a disease or condition recited in this specification. However,the said pharmaceutical compositions may be administered more than onetime, for example from one to four times daily up to a non-limitednumber of days. Specific pharmaceutical compositions are prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound referred to herein above in admixture or otherwiseassociated with a pharmaceutically acceptable carrier or diluent. Formaking those specific pharmaceutical compositions, the activecompound(s) will usually be mixed with a carrier or the diluent, orenclosed or encapsulated in a capsule, sachet, cachet, paper or othersuitable containers or vehicles. The resulting formulations are to beadapted to the mode of administration, i.e. in the forms of tablets,capsules, suppositories, solutions, suspensions or the like. Dosagerecommendations shall be indicated in the prescribers or usersinstructions in order to anticipate dose adjustments depending on theconsidered recipient.

The present invention also relates to a combined preparation forsimultaneous, separate or sequential use comprising at least one virusof the family Paramyxoviridae and at least one multispecific bindingpolypeptide.

A “combined preparation” as referred to in this application preferablycomprises all pharmaceutically active compounds in one preparation sothat all compounds are administered simultaneously and in the same way.

Also preferably, the combined preparation comprises at least twophysically separated preparations for separate administration, whereineach preparation contains at least one pharmaceutically active compound.The latter alternative is preferred in cases where the pharmaceuticallyactive compounds of the combined preparation are administered bydifferent routes, e.g. parenterally and intra-tumorally, e.g. due totheir chemical or physiological properties, or e.g. in cases where theparamyxovirus is administered intra-tumorally and the multispecificbinding polypeptide is administered parenterally.

Preferably, the at least two separated preparations are administeredsimultaneously. This means that the time frames of the administration ofthe preparations overlap.

Also preferred is the sequential administration of the at least twopreparations, wherein the administration of the single preparationsshall occur in time frames which do not overlap, but are selected sothat the at least two pharmaceutically active compounds of thepreparation are present in the body of a subject at least in overlappingtime intervals. Preferably, the at least two preparations areadministered in a time interval of 1 minute, 5 minutes, 15 minutes, 30minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 1 day or 2 days.

Accordingly, the present invention also relates to a method of treatinginappropriate cell proliferation in a subject comprising

a) contacting said subject with a virus of the family Paramyxoviridaeand with a multispecific binding polypeptide according to the presentinvention, andb) thereby, treating inappropriate cell proliferation in a subject.

Preferably, the inappropriate cell proliferation is cancer. Moreover, aswill be understood by the skilled person, contacting a subject may besimultaneous or sequential contacting as specified herein above. As willbe also understood by the skilled person, a subject with a virus of thefamily Paramyxoviridae and with a multispecific binding polypeptide maybe accomplished by contacting said subject with a recombinant virus ofthe family Paramyxoviridae according to the present invention.

Accordingly, the present invention further relates to a method fortreating cancer in a subject afflicted with cancer, comprising

a) contacting said subject with a recombinant virus of the familyParamyxoviridae according to the present invention, andb) thereby, treating cancer in a subject afflicted with cancer.

The methods of treatment of the present invention, preferably, maycomprise steps in addition to those explicitly mentioned above. Forexample, further steps may relate, e.g., to localizing a tumor and/ordiagnosing cancer for step a), or administration of additionalmedication for step b). Moreover, one or more of said steps may beperformed by automated equipment. The method of the present invention,preferably, is an in vivo method of treatment.

The term “treatment” refers to an amelioration of the diseases ordisorders referred to herein or the symptoms accompanied therewith to asignificant extent. Said treating as used herein also includes an entirerestoration of the health with respect to the diseases or disordersreferred to herein. It is to be understood that treating as used inaccordance with the present invention may not be effective in allsubjects to be treated. However, the term shall require that astatistically significant portion of subjects suffering from a diseaseor disorder referred to herein can be successfully treated. Whether aportion is statistically significant can be determined without furtherado by the person skilled in the art using various well known statisticevaluation tools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test etc. Preferredconfidence intervals are at least 90%, at least 95%, at least 97%, atleast 98% or at least 99%. The p-values are, preferably, 0.1, 0.05,0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective forat least 10%, at least 20% at least 50% at least 60%, at least 70%, atleast 80%, or at least 90% of the subjects of a given cohort orpopulation. Preferably, treating cancer is reducing tumor burden in asubject. As will be understood by the skilled person, effectiveness oftreatment of e.g. cancer is dependent on a variety of factors including,e.g. cancer stage and cancer type.

As used herein, the term “subject” relates to a vertebrate. Preferably,the subject is a mammal, more preferably, a mouse, rat, cat, dog,hamster, guinea pig, sheep, goat, pig, cattle, or horse. Still morepreferably, the subject is a primate. Most preferably, the subject is ahuman. Preferably, the subject is afflicted with a disease caused oraggravated by an insufficient response of the immune response of saidsubject, more preferably, the subject is afflicted with cancer.

The term “cancer”, as used herein, relates to a disease of an animal,including man, characterized by uncontrolled growth by a group of bodycells (“cancer cells”). This uncontrolled growth may be accompanied byintrusion into and destruction of surrounding tissue and possibly spreadof cancer cells to other locations in the body. Preferably, alsoincluded by the term cancer is a relapse.

Preferably, the cancer is selected from the list consisting of acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, aids-related lymphoma, anal cancer, appendix cancer,astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer,bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma,carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma,chronic lymphocytic leukemia, chronic myelogenous leukemia, coloncancer, colorectal cancer, craniopharyngioma, endometrial cancer,ependymoblastoma, ependymoma, esophageal cancer, extracranial germ celltumor, extragonadal germ cell tumor, extrahepatic bile duct cancer,fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, gestational trophoblastic tumor, hairy cell leukemia,head and neck cancer, hepatocellular cancer, hodgkin lymphoma,hypopharyngeal cancer, hypothalamic and visual pathway glioma,intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma,medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer,oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarianepithelial cancer, ovarian germ cell tumor, ovarian low malignantpotential tumor, pancreatic cancer, papillomatosis, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sézary syndrome, small cell lung cancer, small intestine cancer,soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer,testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroidcancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer,waldenström macroglobulinemia, and wilms tumor. More preferably, thecancer is a solid cancer, a metastasis, or a relapse thereof. Mostpreferably, the cancer is a tumor derived from malignant melanoma, headand neck cancer, hepatocellular carcinoma, pancreatic carcinoma,prostate cancer, renal cell carcinoma, gastric carcinoma, colorectalcarcinoma, lymphomas or leukemias.

The present invention further relates to an in vitro method foractivating immune cells with antitumor activity in a sample comprisingcancer cells and immune cells, comprising

a) contacting said sample comprising cancer cells and immune cells witha recombinant virus of the family Paramyxoviridae according to thepresent invention, andb) thereby, activating immune cells with antitumor activity comprised insaid sample.

The method for activating immune cells with antitumor activity maycomprise steps in addition to those explicitly mentioned above. Forexample, further steps may relate, e.g., to providing the recombinantvirus of the family Paramyxoviridae for step a), administering furtheractivating compounds, e.g. cytokines, to the immune cells in step b), orseparating immune cells from cancer cells after step b). Moreover, oneor more of said steps may be performed by automated equipment.

The present invention also relates to a recombinant virus of the familyParamyxoviridae according to the present invention for use in medicaltreatment.

Moreover, the present invention relates to a recombinant virus of thefamily Paramyxoviridae for use in treatment of inappropriate cellproliferation.

The term “inappropriate cell proliferation” relates to any proliferationof cells of a subject which is not appropriate to the physiologicalstate of said subject and/or to the tissue context of said cells.Preferably, inappropriate cell proliferation is caused or aggravated byan inhibition or insufficient activation of the immune system, morepreferably inhibition or insufficient activation of T cells. Alsopreferably, inappropriate cell proliferation is cancer.

The present invention further relates to a kit comprising at least therecombinant virus of the family Paramyxoviridae housed in a container.

The term “kit”, as used herein, refers to a collection of theaforementioned components. Preferably, said components are combined withadditional components, preferably within an outer container. The outercontainer, also preferably, comprises instructions for carrying out amethod of the present invention. Examples for such the components of thekit as well as methods for their use have been given in thisspecification. The kit, preferably, contains the aforementionedcomponents in a ready-to-use formulation. Preferably, the kit mayadditionally comprise instructions, e.g., a user's manual for applyingthe recombinant virus of the family Paramyxoviridae with respect to theapplications provided by the methods of the present invention. Detailsare to be found elsewhere in this specification. Additionally, suchuser's manual may provide instructions about correctly using thecomponents of the kit. A user's manual may be provided in paper orelectronic form, e.g., stored on CD or CD ROM. The present inventionalso relates to the use of said kit in any of the methods according tothe present invention.

Summarizing the findings of the present invention, the followingembodiments are preferred:

Embodiment 1

A recombinant virus of the family Paramyxoviridae, comprising at leastone expressible polynucleotide encoding a multispecific bindingpolypeptide, said multispecific binding polypeptide comprising

a) a first binding domain binding to a surface molecule of an immunecell with antitumor activity, preferably a lymphocyte, more preferably aT cell or a dendritic cell, and

b) a second binding domain binding to a tumor-associated antigen.

Embodiment 2

The recombinant virus of the family Paramyxoviridae of embodiment 1,wherein said surface molecule of a T cell is selected from the groupconsisting of CD3, CD2, CD5, CD6, CD9, CD11A, CD25 (IL-2 receptoralpha-chain), CD26, CD28, CD29, CD40L, CD43, CD44, CD45RO, CD45RA,CD45RB, CD47, CD58 (LFA-3), CD69, CD70, CXCR4, CD107a, CD122 (IL-2receptor beta-chain), CD132 (IL-2 receptor gamma-chain), CD134, CD137and CD247, preferably is CD3.

Embodiment 3

The recombinant virus of the family Paramyxoviridae of embodiment 1 or2, wherein said surface molecule of a dendritic cell is selected fromthe group consisting of CD1a/b, CD11c, CD16a, CD40, CD68, CD80, CD83,CD86, IFNAR1 (interferon-alpha/beta receptor), CD119 (interferon-gammareceptor 1), CDB197 (CCR7), CD205 (DEL-205), CD209 (DC-SIGN), and CD227(MUC1).

Embodiment 4

The recombinant virus of the family Paramyxoviridae of embodiment 1 to3, wherein said surface molecule of a natural killer cell is selectedfrom the group consisting of CD16a, NKG2D and NCRs such as NKp30, NKp44and NKp46.

Embodiment 5

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 4, wherein said tumor-associated antigen is atumor-associated antigen exposed on the surface of a tumor cell.

Embodiment 6

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 5, wherein said tumor-associated antigen is selectedfrom the group consisting of androgen receptor (AR), BCL-1,calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial celladhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogenreceptors (mER), FAP HER2/neu, human high molecular weightmelanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21, MOC-52,melan-A/MART-1, melanoma-associated antigen, mucin, OKT9, progesteronereceptor (PGR), prostate specific antigen (PSA), prostate stem cellantigen (PSCA), prostate-specific membrane antigen (PSMA),symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33, preferably iscarcinoembryonic antigen (CEA) or CD20.

Embodiment 7

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 6, wherein said first binding domain is a bindingdomain binding to a surface molecule of a T cell, preferably to CD3.

Embodiment 8

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 7, wherein said multispecific binding polypeptide is abispecific binding polypeptide.

Embodiment 9

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 8, wherein said multispecific binding polypeptide is asecreted multispecific binding polypeptide.

Embodiment 10

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 9, wherein said recombinant virus is a recombinantMorbillivirus, preferably, a recombinant measles virus (MV).

Embodiment 11

The recombinant MV of embodiment 10, wherein said recombinant MV isderived from MV strain Edmonston A or B, preferably vaccine strainEdmonston B.

Embodiment 12

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 11, wherein said first binding domain comprises asingle-chain antibody against CD3.

Embodiment 13

The recombinant virus of the family Paramyxoviridae of embodiment 12,wherein said single-chain antibody against CD3 comprises the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2.

Embodiment 14

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 13, wherein said second binding domain comprises asingle-chain antibody against CEA.

Embodiment 15

The recombinant virus of the family Paramyxoviridae of embodiment 14,wherein said single-chain antibody against CEA comprises the amino acidsequence of SEQ ID NO:3.

Embodiment 16

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 13, wherein said second binding domain comprises asingle-chain antibody against CD20.

Embodiment 17

The recombinant virus of the family Paramyxoviridae of embodiment 16,wherein said single-chain antibody against CD20 comprises the amino acidsequence of SEQ ID NO:4.

Embodiment 18

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 17, wherein the at least one expressible polynucleotideencoding a multispecific binding polypeptide is comprised in apolynucleotide encoding the recombinant virus of the familyParamyxoviridae.

Embodiment 19

The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 18, wherein said multispecific binding polypeptidefurther comprises a cytokine.

Embodiment 20

The recombinant virus of the family Paramyxoviridae of embodiment 19,wherein said cytokine is a cytokine, preferably selected from the listconsisting of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6(IL-6), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocytecolony stimulating factor (G-CSF), granulocyte and macrophage colonystimulating factor (GM-CSF), interferon alpha, interferon betainterferon gamma, and tumor necrosis factor (TNF).

Embodiment 21

A polynucleotide encoding the recombinant virus of the familyParamyxoviridae according to any one of embodiments 1 to 20.

Embodiment 22

The polynucleotide of embodiment 21, wherein said polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:6-9.

Embodiment 23

A medicament comprising the recombinant virus of the familyParamyxoviridae of any one of embodiments 1 to 20 and/or thepolynucleotide of embodiment 21 or 22, and at least onepharmacologically acceptable excipient.

Embodiment 24

A method for treating cancer in a subject afflicted with cancer,comprising

a) contacting said subject with a recombinant virus of the familyParamyxoviridae according of any one of embodiments 1 to 20 and/or witha polynucleotide according to embodiment 21 or 22, and

b) thereby, treating cancer in a subject afflicted with cancer.

Embodiment 25

The method of embodiment 24, wherein said cancer is a solid cancer, ametastasis, or a relapse thereof.

Embodiment 26

The method of embodiment 24 or 25, wherein treating cancer is reducingtumor burden.

Embodiment 27

The method of any one of embodiments 24 to 26, wherein said cancer ismalignant melanoma, head and neck cancer, hepatocellular carcinoma,pancreatic carcinoma, prostate cancer, renal cell carcinoma, gastriccarcinoma, colorectal carcinoma, lymphomas or leukemias.

Embodiment 28

An in vitro method for activating immune cells with antitumor activityin a sample comprising cancer cells and immune cells, comprising

a) contacting said sample comprising cancer cells and immune cells witha recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 20 and/or with a polynucleotide according to embodiment21 or 22, and

b) thereby, activating immune cells with antitumor activity comprised insaid sample.

Embodiment 29

A recombinant virus of the family Paramyxoviridae according to any oneof embodiments 1 to 20 and/or a polynucleotide according to embodiment21 or 22 for use in medical treatment.

Embodiment 30

A recombinant virus of the family Paramyxoviridae according to any oneof embodiments 1 to 20 and/or a polynucleotide according to embodiment21 or 22 for use in treatment of inappropriate cell proliferation.

Embodiment 31

The recombinant virus of the family Paramyxoviridae for use ofembodiment 30, wherein treatment of inappropriate cell proliferation iscancer treatment.

Embodiment 32

Kit comprising at least the recombinant virus of the familyParamyxoviridae according to any one of embodiments 1 to 20 and/or apolynucleotide according to embodiment 21 or 22 housed in a container.

Embodiment 33

A method of treating inappropriate cell proliferation in a subjectcomprising

-   -   a) contacting said subject with a virus of the family        Paramyxoviridae and with a multispecific binding polypeptide        according to the present invention, and    -   b) thereby, treating inappropriate cell proliferation in a        subject.

Embodiment 34

A combined preparation for simultaneous, separate or sequential usecomprising at least one virus of the family Paramyxoviridae and at leastone multispecific binding polypeptide.

Embodiment 35

Use of a virus of the family Paramyxoviridae according of any one ofembodiments 1 to 20, of a polynucleotide according to embodiment 21 or22, of a kit according to embodiment 32, and/or of a combinedpreparation according to embodiment 34, for the manufacture of amedicament for treating disease, preferably for treating inappropriatecell proliferation, more preferably for treating cancer.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic of a viral genome of a multispecific bindingpolypeptide-encoding measles virus (MV-MBP). The 16 kb genomic RNAcomprises 6 genes, encoding 8 proteins. This MeV-MBP additionallyencodes eGFP upstream of N open reading frame (ORF). The transgeneencoding the multispecific binding polypeptide was inserted downstreamof the H ORF. The construct comprises approximately 1,600 bp. The exactnumber of nucleotides is a multiple of six, which is a requirement formeasles viruses. Convenient restriction sites were introduced at theindicated positions to easily transfer the construct to differentvectors and to exchange the TAA-targeting domain. Both, the variabledomains and the scFvs were connected via glycine-serine peptide linkers.The N-terminal HA-tag and C-terminal 6×His-tag are useful for detectionand purification purposes. The actual multispecific binding polypeptidesequence is preceded by a Kozak sequence to enhance translation of thetransgene RNA transcript. In addition, an Igκ-chain leader sequence isfused to the N-terminus of the multispecific binding polypeptide,directing its expression to the secretory pathway.

FIGS. 2a-2c show specific binding of the indicated multispecific bindingpolypeptides to their respective binding domain targets.

FIG. 2a shows specific binding of the indicated multispecific bindingpolypeptides to their respective TAA targets in terms of recombinanthuman protein in a sandwich ELISA format. Polypeptides with the secondbinding domain directed to CEA bind the recombinant CEA full lengthprotein (rCEA). The negative controls mock and non-relevant protein(NRP) (recombinant PD-L1 protein) indicate a specific binding to rCEA.Polypeptides with the second binding domain directed to CD20 show asimilar binding specificity to rCD20. Multispecific binding polypeptideswere detected via anti-HA-tag antibodies.

FIG. 2b shows FACS analyses demonstrating specific binding of theindicated multispecific binding polypeptides with the first bindingdomain directed to human CD3 (hCD3) to peripheral blood mononuclearcells (PBMC) isolated from donor blood.

FIG. 2c shows FACS analyses demonstrating specific binding of theindicated multispecific binding polypeptides with the first bindingdomain directed to murine CD3 (mCD3) on murine splenocytes. Polypeptideswith the first binding domain directed to mCD3 were not found torecognize hCD3 on PBMCs and vice versa, polypeptides with the firstbinding domain directed to hCD3 were not found to recognize mCD3 onmurine splenocytes.

FIGS. 3a-3c show multispecific binding polypeptides-directedcytotoxicity to target cells mediated by PBMCs.

FIG. 3a shows specific killing of MC38 cells expressing the TAA-targetCEA in the presence of PBMCs and multispecific binding polypeptides withthe first binding domain directed to hCD3 and the second binding domaindirected to CEA. Controls with a non-target cell line or a multispecificbinding polypeptide with the second binding domain directed to anirrelevant TAA show no specific tumor cell lysis.

FIG. 3b shows cytotoxicity for multispecific binding polypeptides withthe second binding domain directed to CD20 in a PBMCconcentration-dependent manner.

FIG. 3c shows cytotoxicity for multispecific binding polypeptides withthe second binding domain directed to CD20 in a MBPconcentration-dependent manner.

EXAMPLES

The following Examples shall merely illustrate the invention. They shallnot be construed, whatsoever, to limit the scope of the invention.

Example 1: Production of Virus Encoded Recombinant Multispecific BindingPolypeptides

The amplification of the said pcpNSe-multispecific-binding-polypeptide(pcpNSe-MBP) MV (Edmonston B vaccine strain anti-genome withmultispecific binding polypeptide gene downstream of H open readingframe (ORF) FIG. 1) was performed in NEB 10-beta bacteria, grown in LBmedium (Carl Roth) containing 100 μg/ml ampicillin (Carl Roth). Viralparticles were rescued from pcpNSe plasmids and subsequently propagatedthree times on Vero cells to maximize viral titers. The term rescue ofnegative-strand RNA viruses is known to the skilled person. Transfectionof the plasmids into Vero cells was carried out with FuGENE HD (Promega)according to a standard protocol and cells were incubated at 37° C. forapproximately 65 h. When syncytia had formed, virus particles wereharvested according to the standard procedure. In brief: supernatant wasdiscarded and cells were scraped into fresh medium. Medium was frozen inliquid nitrogen and thawed once, vortexed and centrifuged. Supernatantcontaining the viral particles was aliquoted and stored at −80° C. Forthe production of the multispecific binding polypeptides, 5×10⁶ Verocells were seeded in 15 cm dishes and infected with an MOI of 0.03 in 10ml OptiPRO SFM serum-free medium (Gibco, Invitrogen). Cells were kept at37° C. for approximately 40 h and then transferred to 32° C. foradditional 20 to 25 h. Supernatants were transferred to 50 ml tubes andcentrifuged at 2,000×g, 4° C. for 10 min. Supernatants were passedthrough a 0.22 μm filter (Merck) and multispecific binding polypeptideswere purified via the C-terminal 6×His-tag by affinity chromatographyaccording to standard protocol (Qiagen). His-tagged multispecificbinding polypeptides were eluted with 500 mM imidazole and subsequentlydesalted using centrifugal filters (Amicon, Merck).

Example 2: Characterization of Binding Specificity of RecombinantMultispecific Binding Polypeptides

Specific binding of the multispecific binding polypeptide to human andmurine CD3 and their respective TAA-targets was assessed via FACSanalysis and sandwich ELISA, respectively.

(A) ELISA: 96-well plates (Nunc Maxisorp, Thermo Fisher) were coatedwith 100 μl recombinant human full length CEA (AbD Serotec) or CD20(Abnova) in PBS [1 μg/ml] and kept at 4° C. for at least 16 h. Wellswere washed twice with 200 μl PBS and blocked with 200 μL blockingbuffer (PBS supplemented with 5% FCS and 0.05% Tween20 (Biotium)) for 2h at room temperature. Subsequently wells were washed three times withPBS and incubated with 100 μl sample per well for 2 h at roomtemperature. Wells were washed four times with PBS-T (PBS supplementedwith 0.05% Tween20) and twice with PBS. High affinity anti-HA-biotinantibody (clone BMG-3F10, Roche) was diluted in blocking buffer (1:500).100 μL antibody solution was added to each well and incubated for 45 minat room temperature. Wells were washed five times with PBS-T and twicewith PBS. Streptavidin-horseradish peroxidase (Dianova) was diluted inblocking buffer (1:500) and 100 μL streptavidin solution was added toeach well and incubated for 10 min at room temperature. Wells werewashed seven times with PBS-T and twice with PBS. 100 μl substrate(1-Step Ultra TMB-ELISA, Thermo Fisher) was added to each well andincubated for 3 to 30 min at room temperature. The reaction was stoppedwith 100 μl 2N sulfuric acid per well. Absorbance was measured at 450 nmusing a microplate reader (Infinite M200 Pro).

With the described ELISA procedure, said multispecific bindingpolypeptides with the second binding domain directed to either CEA orCD20 showed specific binding to their respective antigens (FIG. 2a ).The negative controls mock and non-relevant protein (NRP) (recombinantPD-L1 protein) indicate that binding to the respective antigens occursin a specific manner.

(B) FACS: Using flow cytometry, cells were discriminated based on theirsize, structure and surface-expression of particular molecules. Wedetected cell-bound multispecific binding polypeptides via theC-terminal 6×His-tag and anti-His-tag-FITC antibody (DIA920, Dianova,10% in 50 μl). Therefore, we labeled 5×10⁵ human PBMCs from donor bloodor murine splenocytes with multispecific binding polypeptides in FACSbuffer (PBS supplemented with 1 FCS and 0.05% sodium azide, 10% in 50μl, 30 min on ice). To reduce unspecific antibody binding during thefollowing staining procedure, Fc receptors present on cells were blockedusing Kiovig (Baxter) (for human cells) or mouse BD Fc Block (for mouseCD16/CD32) (5% in 50 μl FACS buffer, 5 min on ice). Cells were washedand resuspended in FACS buffer containing antibodies specific forHis-tag, CD3, CD4 and CD8 (BD Biosciences, 5% in 50 μl). After 30 min onice, cells were washed with FACS buffer and resuspended in 500 μl DAPI[0.2 μg/ml]. Cells were washed and resuspended in 200 to 300 μl FACSbuffer and analyzed using an LSR II system (BD Biosciences).

FACS analyses demonstrated specific binding of the multispecific bindingpolypeptides with the first binding domain directed to human CD3 (hCD3)to peripheral blood mononuclear cells (PBMC) isolated from donor blood(FIG. 2b ) and to murine CD3 (mCD3) on murine splenocytes (FIG. 2c ).Polypeptides with the first binding domain directed to mCD3 were notfound to recognize hCD3 on PBMCs and vice versa, polypeptides with thefirst binding domain directed to hCD3 were not found to recognize mCD3on murine splenocytes.

Example 3: Induction of T Cell Effector Function in Resting Human andMurine T Cells by Recombinant Multispecific Binding Polypeptides

We performed lactate dehydrogenase (LDH) release assays to assess thepotential of the multispecific binding polypeptides to induce T celleffector functions, directed to specific tumor cells. 5×10³ target cellswere cocultured with effector T cells at an effector to target cellratio (E:T ratio) of 50:1 or various E:T ratios of 50:1, 25:1, 12:1,6:1, 3:1 and 1:1 on 96-well round-bottom plates in 100 μl RPMI/well intriplicates. Multispecific binding polypeptides were added to each wellat a final concentration of 100 ng/ml or at various concentrations of100 ng/ml, 10 ng/ml, 1 ng/ml, 100 pg/ml, 10 pg/ml and 0 pg/ml.Spontaneous release of LDH from target and effector cells were measuredseparately, as well as maximum LDH release from target cells only usingthe provided lysis solution. Cells were cocultured for 24 h at 37° C.Subsequently plates were centrifuged for 4 min at 250×g and 50 μlsupernatant was transferred to a 96-well flat-bottom plate. LDHconcentration was measured according to the manufacturer's protocol.Tumor-specific T cell-mediated lysis in percent was calculated as:

(experimental release−spontaneous release target cells−spontaneousrelease effector cells)/(maximum release target cells−spontaneousrelease target cells)×100

FIGS. 3a to c demonstrate increased target cell-specific T cell effectorfunction in the presence of the respective multispecific bindingpolypeptides. Tumor cell killing occurred in a T cell- and multispecificbinding polypeptide concentration-dependent manner. The specificitycontrols in FIGS. 3a and 3b demonstrate that neither the binding of themultispecific binding polypeptides to the effector cell alone, nor thecoculture with the respective target cell line in the presence of a CD3binding multispecific binding polypeptide were sufficient to induce Tcell effector functions.

What is claimed is:
 1. A recombinant virus of the familyParamyxoviridae, comprising at least one expressible polynucleotideencoding a multispecific binding polypeptide, said multispecific bindingpolypeptide comprising: a) a first binding domain binding to a surfacemolecule of an immune cell with antitumor activity, wherein said immunecell is a T cell, and wherein said surface molecule is CD3, and b) asecond binding domain binding to a tumor-associated antigen.
 2. Therecombinant virus of the family Paramyxoviridae of claim 1, wherein saidtumor-associated antigen is selected from the group consisting ofandrogen receptor (AR), BCL-1, calprotectin, carcinoembryonic antigen(CEA), EGFRs, epithelial cell adhesion molecule (Ep-CAM), epithelialsialomucin, membrane estrogen receptors (mER), FAP, HER2/neu, human highmolecular weight melanoma-associated antigen (HMW-MAA), IL-6, MOC-1,MOC-21, MOC-52, melan-A/MART-1, melanoma-associated antigen, mucin,OKT9, progesterone receptor (PGR), prostate specific antigen (PSA),prostate stem cell antigen (PSCA), prostate-specific membrane antigen(PSMA), symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33.
 3. Therecombinant virus of the family Paramyxoviridae of claim 1, wherein saidtumor-associated antigen is CEA or CD20
 4. The recombinant virus of thefamily Paramyxoviridae of claim 1, wherein said multispecific bindingpolypeptide is a bispecific binding polypeptide.
 5. The recombinantvirus of the family Paramyxoviridae of claim 1, wherein said recombinantvirus is a recombinant Morbillivirus.
 6. The recombinant virus of thefamily Paramyxoviridae of claim 1, wherein said recombinant virus is arecombinant measles virus (MV).
 7. The recombinant virus of the familyParamyxoviridae of claim 1, wherein said first binding domain comprisesa single-chain antibody against CD3.
 8. The recombinant virus of thefamily Paramyxoviridae of claim 1, wherein said second binding domaincomprises a single-chain antibody against CEA.
 9. The recombinant virusof the family Paramyxoviridae of claim 1, wherein said second bindingdomain comprises a single-chain antibody against CD20.
 10. Therecombinant virus of the family Paramyxoviridae of claim 1, wherein theat least one expressible polynucleotide encoding a multispecific bindingpolypeptide is comprised in a polynucleotide encoding the recombinantvirus of the family Paramyxoviridae.
 11. The recombinant virus of thefamily Paramyxoviridae of claim 1, wherein said multispecific bindingpolypeptide further comprises a cytokine.
 12. A polynucleotide encodingthe recombinant virus of the family Paramyxoviridae according toclaim
 1. 13. A combined preparation for simultaneous, separate orsequential use comprising at least one virus of the familyParamyxoviridae and at least one multispecific binding polypeptide. 14.An in vitro method for treating activating immune cells with antitumoractivity in a sample comprising cancer cells and immune cells,comprising a) contacting said sample comprising cancer cells and immunecells with a recombinant virus of the family Paramyxoviridae of claim 1,and b) thereby, activating immune cells with antitumor activitycomprised in said sample.
 15. A method for treating cancer in a subjectafflicted with cancer, comprising a) contacting said subject with arecombinant virus of the family Paramyxoviridae according to claim 1,and b) thereby, treating cancer in a subject afflicted with cancer. 16.The method of claim 15, wherein step a further comprises contacting saidsubject with a multispecific binding polypeptide.
 17. A kit comprisingat least the recombinant virus of the family Paramyxoviridae accordingto claim 1 housed in a container.